Fig 1.
Resuscitation with AVP restores the mean arterial blood pressure.
(A) Male Long Evans rats were anesthetized with isoflurane (4%) followed by vascular line placement and laparotomy to simulate soft tissue injury. After complete emergence from anesthesia, awake animals were passively bled to a mean arterial pressure (MAP) of 40 ± 5 mmHg. When animals could no longer maintain a MAP of 40 mmHg without fluid (Decompensation), they were given incremental 0.2 cc boluses of lactated Ringer’s (LR) until 40% of the shed volume was returned (Severe Shock). Animals were resuscitated over 60 minutes with 4X the shed volume in LR and either arginine vasopressin (AVP: 0.5 U/kg + 0.03 U/kg/hr) or placebo. Animals were sacrificed at Baseline (Sham n = 9/group), Decompensation (n = 9/group), Severe Shock (n = 9/group), following resuscitation (60R, n = 9/group) and 18 hours post resuscitation (n = 6/group). An additional group of animals were treated with increasing doses of norepinephrine (3 and then 6 ug/kg/min) or epinephrine (1.2 and then 2.4 ug/kg/min). (B) Blood pressure tracings of a representative animal from each group are depicted. Black arrows depict the time vasopressors were initiated, and then increased. (C) The mean arterial blood pressure (MAP) at each time point was recorded. Data were analyzed using one-way ANOVA with a post hoc Tukey’s test. Values are means ± SEM. AVP treated vs. other treatment groups; * p<0.05, ***p<0.001.
Table 1.
The EGTI histology scoring system.
Table 2.
Laboratory values.
Fig 2.
AVP levels decline despite persistent hypotension following hemorrhagic shock.
Pituitary and serum AVP levels were sampled at baseline (Sham), Severe Shock, after 60 minutes of resuscitation (60R) and at 18 hours post resuscitation (n = 6–7 per time point). (A) Posterior pituitary levels (pg/ml ± SEM) of AVP decrease during hemorrhagic shock and were significantly depressed at both 60R and at 18 hours. (B) AVP serum levels (pg/ml ± SEM) appropriately increased but fell significantly following Decompensation. Data were analyzed using one-way ANOVA with a post hoc Tukey’s test. Values are mean ± SEM. Sham vs. other time points; *p<0.05, **p<0.01,***p<0.001, ****p<0.0001. AVP vs. Control; #p<0.05.
Fig 3.
AVP supplementation during hemorrhagic shock is associated with improved mitochondrial function.
(A) Complex I (CI) and (B) Complex II (CII) dependent respiratory capacity in isolated intact mitochondria was assessed by respirometry. Resuscitation with lactated Ringer’s (Control) resulted in impaired CI and CII respiration at 60 minutes and 18 hours. Addition of AVP (0.5u/kg bolus + 0.03u/kg/hr) during resuscitation preserved CI respiration and improved CII dependent respiration. (C). The electron transport from CI to Complex III (CIII) was measured spectrophotometrically in isolated mitochondrial membranes. While control animals demonstrated impaired electron transfer, AVP supplementation preserved CI to CIII electron transfer. (D) The production of radical oxygen species (ROS) in isolated mitochondria was measured using the fluorescent signal from dichlorofluorescein (DCF). The increase in ROS following resuscitation (60R) was mitigated with AVP. (E) Mitochondrial stability was assessed by measuring calcium uptake as a marker of mitochondrial permeability transition. At 18 hours post resuscitation, control mitochondria were less stable than mitochondria isolated from AVP resuscitated animals. N = 6–7 animals per time point. Values are mean ± SEM. Data were analyzed using one-way ANOVA with a post hoc Tukey’s test. *p<0.05 vs. Sham; #p<0.05 Control vs. AVP treated.
Fig 4.
AVP supplementation during hemorrhagic shock is associated with decreased reactive species damage and preserved histologic architecture.
Kidney protein lystates (20μg) were analyzed by SDS-Page using antibodies directed toward 4-hydroxynonenal (HNE) and 3-nitrotyrosine as a measure of oxidative damage. Kidney samples were sectioned, stained with hematoxylin and eosin, and imaged at 10X. (A, B) AVP significantly decreased oxidative damage following resuscitation (60R). (C) AVP also preserved renal architecture with normal glomeruli (depicted by black arrows) observed 18 hours post-resuscitation. N = 6 animals per time point. Values are mean ± SEM. N = 6–7 per time point. Data were analyzed using one-way ANOVA with a post hoc Tukey’s test. Histologic grading was analyzed using a Mann Whiney U test. *p<0.05 vs. Sham; #p<0.05 Control vs. AVP treated.